Polyomaviruses are small, DNA tumor viruses that establish persistent infections in their natural hosts. Several members of the virus family are associated with human pathologies such as Progressive Multifocal Leukoencephalopathy (PML), trichodysplasia spinulosa and Merkel cell carcinoma. Polyomaviruses are one of the first virus family known to encode miRNAs. These polyomaviral miRNAs are located antisense to the early transcripts and hence, mediate the autoregulation of the viral early proteins, the T antigens. There are two major questions in the field of polyomaviral miRNAs – What is the biological significance of this miRNA-mediated autoregulation of the early transcripts? Are there other biological significant targets for these polyomaviral miRNAs? This work addressed these two questions through an evolutionary approach. First, examination of SV40 and JCV variants indicated the high conservation of the miRNAs and their autoregulatory functions. Second, miRNA-mediated autoregulation of the early transcripts is conserved in a newly discovered, evolutionarily divergent viruses, the Bandicoot papillomatosis and Carcinomatosis viruses (BPCVs). Third, by inspecting divergent members of the polyomavirus family, we have shown that some non-human polyomaviruses encode miRNAs, with the function to autoregulate the early transcripts conserved. The conservation of miRNAs both among variants of individual member and across divergent members of the polyomavirus family implies importance. More importantly, a conserved function of autoregulating the early transcript further emphasized the biological relevance of the miRNAs in polyomavirus biology. Yet, the lack of replicative differences between miRNA-expressing and miRNA-null SV40 strains during lytic infections suggests a role for the polyomaviral miRNAs under a different setting, perhaps in the establishment of persistent infection of their natural hosts. This work represents an evolutionary study of polyomaviral miRNAs that has demonstrated the conserved nature of miRNA-mediated autoregulation of the early transcripts among various members of the polyomavirus and polyoma-like virus families. These results have implicated a potential role for the polyomaviral miRNAs in the establishment of persistent infection and raised the possibility of using the JCV miRNAs as potential biomarkers as a non-invasive form of diagnostic for PML. / text

Orientadores: Jose Andres Yunes, Claudia Vianna Maurer Morell, Denise Pontes Cavalcanti / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médica / Made available in DSpace on 2018-08-23T14:33:25Z (GMT). No. of bitstreams: 1 Lucon_DanielleRibeiro_D.pdf: 6878652 bytes, checksum: 72be4de1339573c4cea11e28716998c7 (MD5) Previous issue date: 2013 / Resumo: Crianças com anomalias congênitas possuem um risco significativamente aumentado para desenvolver algum tipo de câncer. Anomalias do sistema nervoso central (SNC) estão associadas à maior incidência de tumores também do SNC. A comparação entre tecido 'anômalo', tecido tumoral e tecido normal pode ajudar na identificação dos genes mais importantes na carcinogênese. microRNAs (miRNAs) são pequenas moléculas que atuam negativamente na expressão gênica e têm papel importante no controle do desenvolvimento, diferenciação, apoptose e proliferação celular. Vários miRNAs são expressos no SNC e são conhecidos por serem dinamicamente regulados durante o neurodesenvolvimento. Recentemente, miRNAs foram associados com tumores e malformações do SNC, como o meduloblastoma (MB) e a anencefalia (AN), respectivamente. Ambos tecidos são de origem neuroectodérmica e embrionária. Neste projeto foram estudados os miRNAs diferencialmente expressos no tecido tumoral de MB desmoplástico de pacientes jovens (1-2 anos) versus cerebelo e no tecido cérebro-vascular de fetos com AN versus córtex frontal. Os controles foram obtidos de tecidos normais provenientes de autópsias de fetos e recém-nascidos. As vias gênica-metabólicas importantes na carcinogênese e morfogênese do perfil de miRNAs de MB e AN foram analisados in silico. No primeiro trabalho, apresentado no segundo capítulo, investigamos o perfil de miRNAs de MB que foi predominantemente baixo expresso (64/84 miRNAs) e regulam genes envolvidos com desenvolvimento e/ou câncer. Muitos dos miRNAs baixo expressos (32/64) foram localizados no lócus cromossômico 14q32 (miRNA 14q32). Possíveis mecanismos da baixa expressão de miRNA 14q32 foram investigados por bancos de dados públicos disponíveis. A expressão do gene receptor de estrógeno gama (ESRRG), um regulador transcricional positivo de alguns miRNAs 14q32, foi encontrada baixo expresso em MB desmoplástico. miR-129-5p (11p11.2/7q32.1), miR-206 (6p12.2) e miR-323-3p (14q32.2) foram escolhidos para estudos funcionais em células DAOY. A super expressão do miR-129-5p usando miRNA mimics diminuiu a proliferação das células DAOY. No segundo trabalho, apresentado no terceiro capítulo, analisamos o perfil de expressão de miRNAs em AN que foi predominantemente super expressos (34/52 miRNAs) e regulam genes envolvidos com defeito do tubo neural e/ou câncer. Dentre estes miRNAs estão os miR-21, 34a/c, 182, 500 cluster. miRNAs importantes no desenvolvimento do cérebro (miR-124, 128, 137, 139) foram encontrados baixo expressos nas amostras de AN. A prospecção dos genes alvos destes miRNAs mostrou que eles desempenham um papel importante durante o desenvolvimento e a diferenciação neural. Por fim, nós comparamos os miRNAs diferencialmente expressos entre MB e AN e identificamos 19 miRNAs em comum (baixo expressos: miR-124, 128, 129*, 129-5p, 138, 138-1*, 138-2*, 139-3p, 490-5p, 650, 770-5p; super expressos: miR-199a-3p, 199b-3p, 199a-5p, 21, 214, 214*, 34a, 574-3p). A maioria destes miRNAs em comum encontrados nas duas patologias fazem parte dos miRNAs mais descritos em câncer e/ou são importantes no desenvolvimento do cérebro. O fato destes miRNAs estarem desregulados em duas condições diferentes (MB e AN) faz pensar que sejam funcionalmente relevantes nestas patologias. Nossos resultados indicam a correlação de assinatura de miRNAs com cada amostra destacando a heterogeneidade molecular e complexidade na sinalização celular regulada por miRNAs, e também revela que o câncer foi a via de sinalização predominante em MB e AN / Abstract: Children with birth defects have a significantly increased risk for developing some type of cancer. Anomalies of central nervous system (CNS) are associated with increased incidence of tumours also from CNS. The comparison between tissue 'anomalous', tumor tissue and normal tissue can help identify genes important in carcinogenesis. microRNAs (miRNAs) are small non-coding RNA molecules that act negatively on gene expression and play an important role in controlling development, differentiation, apoptosis and cell proliferation. Many miRNAs are expressed in CNS and are known to be dynamically regulated in neurodevelopment. Recently, miRNAs have been associated with CNS tumors and malformations, as meduloblastoma (MB) and anencephaly (AN), respectively. Both tissues are from neuroectodermal and embryonic origins. In this project, we studied the miRNAs differential expressed in tumor tissue of desmoplastic MB of young patients (1-2 years) versus cerebellum and cerebrovascular tissue of fetal with AN versus frontal cortex. The normal tissues were obtained from fetal and newborn autopsy. The gene-metabolic pathways important in carcinogenesis and morphogenesis of miRNAs profile of MB and AN were analyzed in silico. In second chapter, we investigated the MB miRNAs profile that were predominantly downregulated (64/84 miRNAs) and regulates genes involved in development and/or cancer. Most downregulated miRNAs (32/64) were found to belong at the 14q32 locus (14q32 miRNA). Possible mechanisms of 14q32 miRNAs downregulation were investigated by the analysis of publicly available gene expression data sets. The expression of estrogen-related receptor-g (ESRRG), a reported positive transcriptional regulator of some 14q32 miRNAs, was found downregulated in desmoplastic MB. miR-129-5p (11p11.2/7q32.1), miR-206 (6p12.2), and miR-323-3p (14q32.2), were chosen for functional studies in DAOY cells. Overexpression of miR-129-5p using mimics decreased DAOY proliferation. In third chapter we investigated the AN miRNAs profile that were predominantly upregulated (34/52 miRNAs) and regulates genes involved with tube neural defects (DTN) and/or cancer. Between these miRNAs are the miR-21, 34a/c, 182, 500 cluster. miRNAs important in brain development (miR-124, 128, 137, 139) were found downregulated in AN samples. Prospecting for target genes of these miRNAs showed that they play an important role during development and neuronal differentiation. Finally, we compare the miRNAs differential expressed between MB and AN and identified 19 miRNAs in common (underexpression: miR-124, 128, 129 *, 129-5p, 138, 138-1 *, 138-2 *, 139 - 3p, 490-5p, 650, 770-5p; overexpression: miR-199a-3p, 3p-199b, 199a-5p, 21, 214, 214 *, 34a, 574-3p). Most common miRNAs found in MB and AN are known to be involved in cancer and/or are important in brain development. The fact that these miRNAs are deregulated in two different conditions (MB and AN) makes one think that they are functionally relevant in these pathologies. Our results indicate the correlation of miRNAs signature with each sample highlighting the molecular heterogeneity and cellular signaling complexity regulated by miRNAs, and also reveals that the cancer was the predominant signaling pathway in MB and AN / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Orientador: Flavia Lombardi Lopes / Coorientador: Marcia Marinho / Banca: Juliana Regina Peiro / Banca: Jaqueline Carvalho de Oliveira / Banca: kathlenn Liezbeth de Oliveira da Silva / Resumo: Leptospirose é considerada uma zoonose de importância global causada pela bactéria Leptospira spp. A incidência da doença chega a um milhão de casos severos no mundo, atingindo uma mortalidade de 60.000 óbitos/ano. Apesar de considerados avanços na pesquisa sobre a doença, há muito ainda a se descobrir sobre sua patogenicidade. A influência de mecanismos epigenéticos em processos patológicos, particularmente da regulação pós-transcricional mediada por RNAs não-codificadores, é fundamental. Uma gama de estudos vem relatando a influência desses mecanismos na resposta imune do hospedeiro em uma variedade de infecções bacterianas. O objetivo desse estudo foi identificar e caracterizar, pela primeira vez, a influência de miRNAs na regulação da resposta de macrófagos frente a infecção por Leptospira. Foi realizado um microtranscriptoma, através da técnica de microarranjo, em macrófafgos murinos J774A.1 desafiados por cepa virulenta, atenuada e saprófita de Leptospira após 8h de infecção. Após análise dos dados, identificamos 29 miRNAs modulados pela infecção comparados ao controle, com fold change ± 1.5 e p-valor<0,01. Na análise de enriquecimento funcional, encontramos um grande número de alvos para os miRNAs modulados participando em processos-chave da resposta imune. Podemos sugerir que a regulação pós-transcricional por miRNAs possui papel na resposta do hospedeiro em infecção por Leptospirose, e que essa resposta é dependente da espécie, bem como da virulência da bactéria. / Abstract: Leptospirosis is a bacterial zoonosis of global importance, caused by Leptospira. The incidence of disease leading to one million severe cases and 60,000 deaths per year. Despite considerable advances in research, much is yet to be discovered about disease pathogenicity. The influence of epigenetic mechanisms, particularly RNA-mediated post-transcriptional regulation of host immune response has been proven vital following a variety of bacterial infections. The aim of this study was to identify and characterize, for the first time, the influence of miRNAs on the regulation of the immune response to Leptospira infection. We examined the microtranscriptome, trough microarray technique, of macrophages J774A.1 following an 8h infection with virulent, attenuated and saprophyte strains of Leptospira. Microarray analysis revealed that 29 miRNAs were de-regulated by the Leptospira strains compared to control with fold change ±1.5 and p-value <0.01. Enrichment analyses of the targets of these differentially expressed miRNAs suggest that several processes involved in immune response are directly regulated by miRNAs. We suggest that posttranscriptional regulation by miRNAs may play a role in host response to infection in leptospirosis, and that this response is dependent on species and bacterial virulence. / Doutor

RNAs endógenos competitivos (ceRNAs) são transcritos naturais que atuam como esponjas endógenas de miRNAs, modulando a ação de miRNAs sobre mRNAs-alvo. RNA circular (circRNA) é uma dentre as várias classes de ceRNA. circRNAs são produzidos a partir de um processo chamado backsplicing. CircRNAs já foram identificados em diversos eucariotos; no entanto, nas plantas, ainda não foi demonstrado se estes são capazes de atuar como esponjas eficazes de miRNAs. Dados públicos de RNAseq de bibliotecas de imunoprecipitação de Argonauta (AGO-IP) a partir de flores de Arabidopsis thaliana foram utilizados em um método multi-comparativo de análises de bioinformática para identificar potenciais RNAs circulares. Utilizando cinco diferentes métodos, foram preditos de 15 a 27812 circRNAs com pelo menos dois reads na junção do backsplicing. Posteriormente, a plataforma psRNAtarget foi utilizada para discriminar os circRNAs com sítios de ligação de miRNAs, que potencialmente possam estar atuando como esponjas. Como a AGO forma um complexo ternário com miRNA e mRNA-alvo, também foram comparadas as contagens de alvos em bibliotecas AGO-IP e controle, demonstrando que os mRNAs-alvo desses miRNAs também estão enriquecidos nas bibliotecas AGO-IP. Neste trabalho, foram encontrados dois prováveis circRNAs que podem potencialmente funcionar como esponjas de miRNA. Esse achado contribui para um melhor entendimento desta nova forma de regulação transcricional e pós-transcricional em plantas, ampliando o conhecimento e aplicações do tema. / Competing endogenous RNAs (ceRNAs) are natural transcripts that act as endogenous sponges of miRNAs, modulating the action of miRNAs on mRNAs targets. Circular RNA (circRNA) is one among the various classes of ceRNA. They are produced from a process called backsplicing. CircRNAs have been identified in several eukaryotes; however, in plants, it has not yet been demonstrated whether they are able to act as effective sponges for miRNAs. Public RNAseq data from Argonaute immunoprecipitation libraries (AGO-IP) from Arabidopsis thaliana flowers were used with a multi-comparative method of bioinformatics analyzes to identify putative circular RNAs. Using five different methods, 15 to 27812 circRNAs with at least two reads at the backsplicing junction were predicted. Subsequently, the psRNAtarget platform was used to discriminate circRNAs harboring miRNA binding sites, which could potentially be acting as sponges. Identities and amounts of mRNAs present in AGO-IP and control libraries were quantified, as AGO can also form a ternary complex among miRNA and their target mRNAs. It showed that target mRNAs from circRNA:miRNA pairs were also enriched in the AGO-IP libraries. Two circRNAs were positively identified, corroborating their action as potential miRNA sponges. This finding leads to a better understanding of a new form of transcription and post-transcription regulation in plants, increasing the knowledge and applications of the topic.

在不同的模型體系和疾病microRNA-199a (miR-199a)的功能和基因表達都不盡相同而且相當複雜。它在人基因組中有個表達位點，分別是位於19 號染色體的miR-199a-1 和1 號染色體的miR-199a-2。這個位點可以產生相同的miRNA (miR-199a-3p 和miR-199a-5p)。造成miR-199a不同表達和功能的原因尚未清楚。但是，很有可能是由於個位點不同的調節機制和miR-199a 在不同的細胞和組織中可變的作用对象。在睾丸生殖細胞腫瘤(簡稱睾丸癌)和膠質母細胞瘤(簡稱膠質瘤)中研究miR-199a 的啟動子甲基化發現，在睾丸癌中miR-199a-1 和-2 共同的啟動子過甲基化導致miR-199a 表達低。而在膠質瘤中，只有miR-199a-2，而不是miR-199a-1 的啟動子低甲基化與miR-199a 的高表達相關。除啟動子甲基化，在幹細胞分化過程中升高表達的miR-199a 被證實是通過轉因子Twist1 的調控；循环通Twist1-miR-199a-5p-HIF1α控制miR-199a-5p 促進成骨分化的作用。miR-199a 的功能是通過其下游目標 (靶基因) 而成的。因此, 目標分子的功能控制miR-199a 在同的模型體系和疾病中的作用。我應用基因組學和蛋白組學的方法尋找miR-199a-5p 在睾丸癌中的下游目標(靶基因)。MAFB 被指出和證明是miR-199a-5p 在睾丸癌中的靶基因，而miR-199a 抗腫瘤細胞增殖活性的作用是通過MAFB 發揮的。通過研究miR-199a 在用系統中的調控機制和作用目標，我希望用miR-199a 作為模型明miRNA 生物學的複雜多樣性。 / microRNA-199a (miR-199a) has been shown to have diverse biological functions and behave quite differently in different physiological systems and diseases. It is encoded by two loci in the human genome, miR-199a-1 in chromosome 19 and miR-199a-2 in chromosome 1. Both loci give rise to the same miRNAs (miR-199a-5p and miR-199a-3p). The underlying mechanism responsible for the diverse action of the miRNA is not clear. However, it is likely contributed by differential regulation of the two genomic loci and variable targets of the miRNA in different cells and tissues. Studies on the promoter methylation of miR-199a in testicular germ cell tumors (TGCTs) and glioblastomas (gliomas) demonstrated that hypermethylation of both loci of miR- 199a resulted in its reduced expression in TGCTs, while hypomethylation of miR-199a-2 but not -1 in gliomas might lead to its elevated expression. In addition to DNA methylation, the functions of transcription factors in controlling the expression of miR-199a through a Twist1- miR-199a-5p-HIF1α cyclic pathway were demonstrated to play significant roles during me nchymal stem cell differentiation. The functions of miR-199a are mediated by the actions of its downstream targets. Both genomic and proteomic approaches were applied to study the targets of miR-199a-5p in TGCTs. A putative target of miRNA-199a-5p, MAFB, was identified and confirmed to be responsible for the anti-tumor proliferation activity of the microRNA. By studying the mechanisms that control the expressions of miR-199a and its various downstream targets in different systems, it is hoped that miR-199a could be used as a model to illustrate the complexity of miRNA biology. / Detailed summary in vernacular field only. / Gu, Shen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 144-163). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.II / Acknowledgements --- p.III / Abbreviations --- p.VIII / Chapter 1 --- p.1 / Chapter 1.1 --- miRNAs - small yet powerful molecules --- p.2 / Chapter 1.1.1 --- Biogenesis and processing of miRNA --- p.2 / Chapter 1.1.2 --- Detection of miRNA expression --- p.7 / Chapter 1.1.3 --- Identification of putative miRNA targets --- p.11 / Chapter 1.2 --- miR-199a - an example revealing the complexity of the miRNA world --- p.19 / Chapter 1.2.1 --- miR-199a - standing out among thousands --- p.19 / Chapter 1.2.2 --- Regulation of miR-199a expression --- p.20 / Chapter 1.2.3 --- miR-199a in carcinogenesis --- p.24 / Chapter 1.2.4 --- miR-199a in cardiogenesis --- p.33 / Chapter 1.2.5 --- miR-199a in stem cell differentiation and embryo development --- p.34 / Chapter 1.2.6 --- Other functions of miR-199a --- p.35 / Chapter 1.3 --- Project overview --- p.38 / Chapter 2 --- p.39 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.2 --- Materials and methods --- p.42 / Chapter 2.3 --- Results --- p.49 / Chapter 2.3.1 --- Dysregulation of miR-199a in tumors were controlled by DNA methylation --- p.49 / Chapter 2.3.2 --- Transcriptome changes upon miR-199a-5p in TGCTs --- p.58 / Chapter 2.3.3 --- Identification of MAFB as a putative target of miR-199a-5p in TGCTs --- p.63 / Chapter 2.3.4 --- MAFB is highly expressed in malignant testicular tumor and negatively correlated with miR-199a-5p --- p.67 / Chapter 2.3.5 --- MAFB knockdown suppresses tumor cell growth in vitro --- p.69 / Chapter 2.4 --- Discussion --- p.70 / Chapter 3 --- p.75 / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Materials and methods --- p.81 / Chapter 3.3 --- Results --- p.86 / Chapter 3.3.1 --- Expression of miR-199a during MSC osteogenesis --- p.86 / Chapter 3.3.2 --- Functions of miR-199a on osteogenesis of hMSCs --- p.90 / Chapter 3.3.3 --- Involvement of HIF1α, Twist1 and miR-199a-5p during osteogenesis of hMSC at early stage and late stage of differentiation --- p.94 / Chapter 3.3.4 --- Up-regulation of miR-199a-5p was related to hypoxia enhanced osteogenesis at early stage --- p.99 / Chapter 3.3.5 --- miR-199a-5p inhibited HIF1α-Twist1 pathway to increase osteogenesis at late stage --- p.103 / Chapter 3.4 --- Discussion --- p.107 / Chapter 4 --- p.111 / Chapter 4.1 --- Overview of the project --- p.112 / Chapter 4.2 --- Summary and conclusion --- p.115 / Chapter 4.3 --- Future work --- p.116 / Chapter Supplementary Materials --- p.117 / Chapter Supplementary Table 2.1 --- p.118 / Chapter Supplementary Table 2.2 --- p.129 / Chapter Supplementary Table 2.3 --- p.142 / References --- p.144

Orientador: Patricia Pintor dos Reis / Resumo: Introdução: O carcinoma de pâncreas apresenta altas taxas de mortalidade, geralmente associadas ao diagnóstico tardio da doença. MicroRNAs (miRNAs) são importantes reguladores da expressão gênica e têm sido apontados como biomarcadores com valor diagnóstico, prognóstico no câncer. Objetivos: (1) identificar níveis de expressão de miRNAs em tumores de pacientes com câncer de pâncreas; (2) identificar, “in silico”, genes-alvo dos miRNAs e vias moleculares envolvendo miRNAs e genes-alvo correspondentes; (3) realizar estudos funcionais “in vitro” para demonstrar experimentalmente os efeitos da desregulação de miRNAs nos potenciais de migração e invasão celulares. Materiais e Métodos: Foram incluídas 19 amostras de adenocarcinoma ductal pancreático (PDAC) e 6 de adenocarcinoma de ampola de Vater (AMP), pareadas com tecido histologicamente normal. Além disso, 3 linhagens celulares de carcinoma pancreático (BxPC-3, Capan-2 e Panc-1) foram analisadas quanto ao seu perfil global de expressão de miRNAs. Em todas as amostras, o perfil de expressão de miRNAs foi determinado utilizando a plataforma GeneChip® 4.0 miRNAarrays. A validação “in silico” utilizou dados de transcriptoma do The CancerGenome Atlas (TCGA) (N=178 carcinomas pancreáticos e 4 tecidos histologicamente normais dos mesmos pacientes). A linhagem Panc-1 foi testada em ensaios de migração e invasão antes e depois da expressão dos miRNAs miR-148a-3p e miR-216b-5p por meio de moléculas miméticas. Análises computacionais utili... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Pancreatic carcinoma is associated with high mortality rates, usually due to late disease diagnosis. MicroRNAs (miRNAs) are important gene expression regulators and have been identified as biomarkers with diagnostic, prognostic and therapeutic value in cancer. Objectives: (1) to identify miRNA expression levels in tumors of patients with pancreatic cancer; (2) to identify, in silico, miRNA target genes and molecular pathways involving miRNAs and corresponding target genes; (3) to perform in vitro functional studies to experimentally demonstrate the effects of miRNAdysregulation on cell migration and invasion. Materials and Methods: We included 19 pancreatic ductal adenocarcinoma (PDAC) and 6 adenocarcinoma of Vater ampulla (AMP) samples, paired with histologically normal tissues. In addition, 3 pancreatic carcinoma cell lines (BxPC-3, Capan-2 and Panc-1) were analyzed for their global miRNA expression profile. miRNA expression profiles were determined using the GeneChip® 4.0 miRNA arrays platform. In silico validation used transcriptome data from The Cancer Genome Atlas (TCGA) (N = 178 pancreatic carcinomas and 4 histologically normal tissues from the same patients). The Panc-1 cell line was used for migration and invasion assays before and after miR-148a-3p and miR-216b-5p expression by mimetic molecules. Computational analyzes used bioinformatics algorithms to identify target genes regulated by miRNAs (miRNA Data Integration Portal, mirDIP) and molecular pathw... (Complete abstract click electronic access below) / Doutor

Schistosomiasis caused by the parasitic helminth Schistosoma mansoni is a major health problem in tropical and subtropical regions. Its detection is crucial for patient management, evaluation of treatment, and monitoring of disease transmission, thus the development of novel diagnostic assays is of immense importance. The pathology of S.mansoni infection is characterised by formation of granulomatous lesions, hepatic fibrosis and portal hypertension. Liver fibrosis itself, regardless of the causative agent is an important health problem. The fact that there is no treatment for hepatic fibrosis other than transplantation in end stage liver failure emphasizes the importance of investigating the molecular basis of fibrogenesis and development of better therapeutic tools. MicroRNAs (miRNAs) are a class of short non-coding RNA that play important roles in disease processes in animals. Several miRNAs have been implicated in hepatic fibrogenesis; however, the expression profile and the role of miRNAs in S. mansoni infection are yet to be determined. This thesis focuses on the characterization of miRNAs in the liver and serum of mice during S.mansoni infection in order to determine their therapeutic and diagnostic potential in this disease. Profiling of miRNA expression in the liver of mice infected with S.mansoni revealed a set of mouse miRNAs that were differentially expressed in infected compared to naïve mice including miR-199a-3p, miR-199a-5p, miR-214 and miR-21, which have previously been associated with liver fibrosis in other settings. Further, inhibition of one of the up-regulated miRNAs, miR-199a-3p, in the liver upon S.mansoni infection resulted in reduced levels of collagen and other fibrosis related genes. Our results are consistent with a model where miRNA inhibition influences the clearance or reversion of hepatic stellate cells (HSCs) from a “myofibroblast-like” to an inactivated state. Thus, these results suggest miR-199a-3p inhibitor as potential therapeutic in treatment of liver fibrosis. miRNAs have been shown to be altered in disease process and are present in body fluids in a stable form, indicating that they can be used as novel diagnostic biomarkers. Studies of miRNAs in the circulation revealed that 5 of the mouse miRNAs altered in the liver were also significantly elevated in serum by 12 weeks post-infection. Sequencing of small RNAs from serum confirmed the presence of these miRNAs and further revealed 11 parasite-derived miRNAs that were detectable by 8 weeks post infection. Analysis of host and parasite miRNA abundance by qRTPCR was extended to the serum of patients from low- and high-infection sites in Zimbabwe and Uganda. The host-derived miRNAs failed to distinguish uninfected from infected individuals. However, analysis of three of the parasite-derived miRNAs (miR-277, miR-3479-3p and bantam) could detect infected individuals from low- and high-infection intensity sites with specificity/sensitivity values of 89%/80% and 80%/90%, respectively. Moreover, sequencing of small RNAs from serum revealed that specific tRNA fragments of 29-33 nt exist in mouse serum and occur at a >10 fold higher copy number than known microRNAs. The tRNA fragments appear to be the product of a specific cleavage event near the anti-codon loop, which has previously been associated with oxidative stress inside cells. We detected a clear bias in the abundance of 5’ versus 3’ products of tRNAGly(GCC), indicating specificity in the mechanism of stabilization of these products following cleavage. Our findings suggest that these specific tRNA cleavage products are either generated in, or exported to, serum and are protected against serum RNase activity by protein rather than encapsulation within vesicles. Further work is necessary to investigate the role and potential involvement of tRNA halves in cell-to-cell communication in oxidative stress induced by S.mansoni infection. In summary, this thesis reveals three major findings, which provide an important base for further studies of the role of miRNAs in S.mansoni infection and liver fibrosis. Firstly, it characterises host miRNAs dysregulated during S.mansoni infection in the liver and identifies a miR-199a-3p inhibitor as a potential anti-fibrotic agent. Secondly, it identifies parasite-derived miRNAs as novel markers of S. mansoni infection in the serum of both mice and humans, with the potential to be used with existing techniques to improve S.mansoni diagnosis. Lastly, it identifies and characterises novel tRNA-derived fragments in the serum, whose properties as biomarkers awaits further characterisation.

O complexo Cryptococcus possui 37 espécies descritas, das quais se destacam C. neoformans e C. gattii devido a sua capacidade de causar doença tanto em humanos como animais. A fim de se prevenir de organismos patogênicos, o hospedeiro pode reduzir a disponibilidade de nutrientes em um processo conhecido como imunidade nutricional. Além disso, é bem descrito o papel central de metais essenciais em relação à expressão dos fatores de virulência nas espécies patogênicas do gênero Cryptococcus. MicroRNAs (miRNAs) são importantes mediadores da expressão gênica em resposta a estresses, incluindo privação de metais ou toxicidade causada pelos mesmos. Entretanto, pouco é conhecido sobre expressão de miRNAs ou miRNAs-like em C. gattii. Portanto, o objetivo desde trabalho foi determinar o perfil miRNAs ou miRNAs-like produzidos por C. gattii durante privação de ferro e privação de zinco. Células de C. gattii R265 foram cultivadas em três condições: privação de zinco, privação de ferro e condição controle. As sequências dos pequenos RNAs foram obtidas por RNA-Seq. A fim de identificar miRNAs em C. gattii, as sequências não redundantes foram alinhadas às sequências dos genomas das linhagens de C. gattii R265 e WM276. Foi realizada a predição de miRNAs ou microRNA-like com ferramentas de bioinformática que resultou em 31 possíveis miRNAs na linhagem R265 e 26 na linhagem WM276, respectivamente. Análises in silico da expressão diferencial dos miRNAs mostraram que a maioria está presente nas três condições, ao passo que apenas dois estão presentes apenas nas condições de privação de metais. Desta forma, estes miRNAs-like podem estar exercendo algum papel importante na homeostase de metais em resposta a condições que mimetizam o ambiente de infecção. Visto que a linhagem R265 de C. gattii não possui um gene codificador da proteína Argonauta, a qual desempenha um papel central na via de RNA de interferência (RNAi), esta via possivelmente não seria responsável pelo silenciamento de outros genes. Levanta-se a hipótese que estes miRNAs-like poderiam ser exportados da célula e, então, modular a expressão gênica no hospedeiro. Esta análise foi iniciada pela avaliação de possíveis alvos expressos pelos genomas de camundongo e humano. Alguns destes alvos mostraram relação com a resposta imune nos hospedeiros. Esta identificação deve esclarecer o papel desenvolvido por miRNAs na regulação da expressão gênica na interação patógeno-hospedeiro. / The Cryptococcus complex has 37 species described, from which C. neoformans and C. gattii stand out for their ability to cause disease in humans and animals. To prevent infection with pathogenic microorganisms, host may reduce the availability of nutrients in a process known as nutritional immunity. Furthermore, it is well described that essential metals have central roles related to the regulation of expression of virulence factors in pathogenic Cryptococcus species. MicroRNAs (miRNAs) are important mediators of gene expression in response to several stresses, including metal deprivation or toxicity. However, little is known about microRNAs or miRNAs-like expression in C. gattii. Therefore, the aim of this work was to profile miRNAs produced by C. gattii during iron and zinc deprivation. C. gattii R265 cells were incubated in three conditions: zinc deprivation, iron deprivation and control condition. The sequences of small RNAs were obtained by RNA-Seq. To identify miRNAs or miRNAs-like in C. gattii, non-redundant reads were aligned to sequences of C. gattii R265 and WM276 strains genomes. MiRNAs prediction was and resulted in 31 possible miRNAs in R265 strain and 26 in WM276 strain, respectively. In silico analysis of miRNAs differential expression showed that the majority of them are present in the three conditions, while two are present only in metal deprivation. Thereby, these miRNAs-like may be exerting some important role at metal homeostasis in response to infection environment mimicry conditions. As the C. gattii R265 strain does not have an Argonaut encoding gene, the protein that plays a central role in the RNA interference (RNAi) pathway, we assumed that probably this pathway would not be responsible for silencing of others genes. We hypothesized that these miRNAs-like could be exported and modulate the gene expression in host cells. This analysis was initialized by evaluating the potential targets expressed by human and mouse genome. Some of these targets were related to immune response in the host. This identification should clarify the role played by miRNAs in the regulation of gene expression in the host-pathogen interaction.

Orientador: Flávia Lombardi Lopes / Resumo: Os microRNAs (miRNAs) são pequenos RNAs não-codificadores (20-22 nucleotídeos) que exercem uma função de controle pós-transcricional em RNA mensageiro (RNAm), regulando a produção de proteínas. Variações genéticas, como os polimorfismos de nucleotídeo único (SNPs), quando estão presentes em sequências de miRNA, podem alterar o controle pós-transcricional, baseado em similaridade aos seus respectivos alvos. Na produção de suínos, a identificação de fenótipos, como crescimento e características da carne, é vital. Nosso objetivo foi identificar SNPs em sequências genômicas que dão origem a miRNAs, bem como investigar, in silico, possíveis alterações na regulação de alvos que poderiam estar relacionados a fenótipos de produção em suínos. A montagem do assembly Sscrofa10.2 foi utilizada como genoma de referência para a localização dos SNPs e dos miRNAs descritos em suínos. Utilizando um script desenvolvido em Python, foi possível localizar 86 SNPs em sequencias miRNAs maduros. Para a predição dos genes alvos, foram utilizados sequência do 3´UTR de suínos com a adaptação do pacote Mirmap. Descobrimos que 55 das sequências de miRNA geraram mais de 28.570 genes alvos, indicando a criação de novos miRNAs Interagindo com RNAm alvos. Nossos resultados indicam que SNPs afetam a predição de alvos para miRNAs em suínos. / Abstract: The microRNAs (miRNAs) are small non-coding RNAs (20-22 nucleotides) that exert a post-transcriptional control function in messenger RNA (mRNA), regulating the production of proteins. Genetic variations, such as single nucleotide polymorphisms (SNPs), when present in miRNA sequences, may change post-transcriptional control, based on similarity to their target genes. Pig production, identification of phenotypes, growth and meat characteristics is vital. Other in which SNPs in genetic sequences that give from miNNAs, as well as investigate, in silicon, changes in the data of alves, which are behaviert on a phenotypes of production in swines. The assembly of the Sscrofa10.2 assembly was used as a reference reference for the location of SNPs and panel miRNAs in swine. Using a script deployed in Python, it was possible to locate 86 SNPs in mature miRNAs sequences. For a prediction of the target genes, the 3'UTR of pigs were completed with an adaptation of the Mirmap package. Discoveries that 55 of the miRNA sequences generated more than 28,570 target genes, pointing to a new creation of miRNAs interacting with mRNA targets. Cloister all SNPs for a target prediction for miRNAs in pigs. / Mestre

Diversos trabalhos demonstram que a resposta das plantas ao alumínio é complexa. Porém, nenhum trabalho caracterizou o envolvimento de microRNAs nesta resposta. Parte deste trabalho visou caracterizar o perfil de expressão de diferentes famílias de microRNAs em resposta ao alumínio comparando-se as raízes de plantas de arroz japonica e indica. De um total de dezesseis microRNAs diferencialmente expressos, treze microRNAs tiveram a expressão reduzida e outros seis tiveram a expressão aumentada nas raízes de plantas de arroz japonica tratadas com 450 M de AlCl3 após 8h. Nas plantas de arroz indica tratadas nas mesmas condições, nove microRNAs foram detectados como diferencialmente expressos. Destes, quatro tiveram a expressão aumentada e os outros cinco tiveram a expressão reduzida. Por RT-qPCR foram confirmados dois alvos do miR528. Um dos alvos, o gene L-Ascorbato Oxidase está relacionado com a regulação da divisão celular. Sugere-se que a regulação pelo miR528 pode estar de acordo com a inibição do desenvolvimento das raízes em resposta ao alumínio em plantas sensíveis. Estes resultados demonstram que a resposta dos microRNAs ao tratamento com alumínio também é complexa, pois vários microRNAs, que provavelmente regulam alvos distintos tiveram sua expressão modulada após o tratamento das plantas. Já foi demonstrado que o desenvolvimento de raízes laterais sofre regulação por microRNAs em Arabidopsis. A outra parte deste trabalho visou caracterizar funcionalmente o miR164 e a expressão espacial de diferentes membros da família miR164 em raízes de plantas de arroz. Plantas superexpressando o miR164 apresentaram as raízes laterais reduzidas em comparação com as plantas não transformadas. Interessantemente, a análise por RT-qPCR de dois alvos do miR164 revelaram resultados inversos. A análise das plantas contendo os promotores de três genes da família miR164 fusionados ao gene repórter GUS revelou uma sobreposição da expressão espacial no órgão. Os microRNAs miR164a e miR164d localizam-se nas raízes laterais, e o miR164f está localizado nas raízes laterais e também na raiz primária. Porém, cortes transversais das raízes demonstraram que os miR164a e o miR164f localizam-se na endoderme e no estelo, respectivamente. Uma análise in silico das seqüências dos promotores dos seis membros da família miR164 e de seus respectivos alvos revelou a presença de motivos de DNA provavelmente responsivos a fatores de transcrição envolvidos no desenvolvimento de meristemas primários. Com base nestes resultados, sugere-se que os microRNAs miR164a e miR164f devem estar regulando seus alvos em diferentes tecidos da raiz. Este resultado está de acordo com o desenvolvimento inicial das raízes laterais, pois estas se originam do periciclo componente do estelo. Os microRNAs têm função crucial ao longo do desenvolvimento e em resposta a estresses abióticos. O papel regulatório dos microRNAs reside na complexidade e diversidade das respostas a estresses abióticos e ao desenvolvimento, visto que diversos microRNAs, que provavelmente regulam distintos alvos, estão envolvidos em redes complexas na regulação da expressão gênica. / Previous works showed that the response to aluminum (Al) in plants is complex. However, at present, there is no data regarding microRNA expression in this response. Part of this work aimed to characterize the expression profile of different families of microRNAs in response to Al comparing roots of japonica and indica roots. From sixteen microRNAs differentially expressed, thirteen were down-regulated and six were upregulated in japonica rice roots treated with 450 M of AlCl3 after 8h. For the indica rice roots under the same conditions, nine microRNAs were differentially expressed. From these microRNAs, four were up-regulated and five were down-regulated. Two miR528 targets were confirmed by RT-qPCR. One of the targets is the L-AScorbate oxidase gene, which regulate cell divisions. These results help explaining rice root inhibition under Al teatment in sensitive plants. Our results suggest that microRNA response to Al is also complex, because several microRNAs that had their expression modulated probably regulate distinct target genes. It is known that lateral root development is regulated by microRNAs in Arabidopsis. The other part of this work was to characterize the miR164 function and the spatial expression of different members of the miR164 family in rice roots. Plantas overexpressing the miR164 had less lateral roots when compared to the non-transformed plants. Interestingly, the expression by RT-qPCR of two miR164 target was inverse in the miR164 overexpressing plants. The spatial expression analysis of different members of the miR164 family revealed overlapping domains. MicroRNAs miR164a and miR164d localized in the lateral roots, and miR164 is localized in lateral roots and also in primary roots. However, hand-sectioning of the miR164a and miR164f GUS fusion plants demonstrate that miR164a is expressed in the endodermis and miR164f is expressed in the stele. An in silico analysis of promoter sequences of all miR164 genes revealed the presence of DNA motifs probably responsive to transcription factors involved in the development of primary meristems. Base on these results, we suggest that different members of the miR164 family are regulating their targets in different tissues of rice roots. These results are in agreement with the initial development of lateral roots originating from the pericycle cells. The key of the regulatory role of microRNAs in response to abiotic stresses and during development brought complexity to the regulatory networks of gene expression.